Objective: Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis.
Methods: Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group ( n=12) and DBM group ( n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67].
Results: Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly ( t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD ( P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM ( P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity ( P<0.05), and the number of CD31 hiEmcn hi cells increased significantly ( P<0.05).
Conclusion: DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.
目的: 基于选择性细胞滞留（selective cell retention，SCR）技术中的细胞与细胞外基质黏附理论，拟设计一种简化多肽表面修饰脱钙骨基质（demineralized bone matrix，DBM），以期能同时促进骨再生与血管生成。.
方法: 选择层粘连蛋白（laminin，LN）α4 链核心区域功能肽（LNα4）、环状 RGD（cyclic RGDfK，cRGD）和带胶原结构域肽（collagen-binding domain，CBD），固相合成 CBD-LNα4-cRGD 多肽，表面修饰于 DBM，构建 DBM/CBD-LNα4-cRGD（以下简称 DBM/LN）支架材料。观测 DBM/LN 形态特征、CBD-LNα4-cRGD 表面修饰 DBM 的稳定性以及 CBD-LNα4-cRGD 表面修饰对小鼠血管内皮祖细胞（endothelial progenitor cells，EPCs）黏附、增殖以及管腔形成的影响，Western blot 验证其影响 EPCs 血管生成的分子机制。然后，取 24 只 10 周龄雄性 C57 小鼠制备 2 mm 长股骨缺损模型，分别采用经 SCR 技术处理的 DBM/LN（DBM/LN 组 ， n=12）和 DBM（DBM 组， n=12）修复骨缺损。术后 8 周，行影像学检查、血管造影、组织学及免疫荧光染色［CD31、内皮黏蛋白（endomucin，Emcn）、Ki67］观察，评估 DBM/LN 促进血管生成和骨再生能力。.
结果: 材料相关检测显示，与 DBM 相比，DBM/LN 表面更粗糙，但孔径差异无统计学意义（ t=0.218， P=0.835）；经 SCR 技术处理后，荧光强度提示 DBM/LN 支架稳定有效；与 DBM 相比，CBD-LNα4-cRGD 表面修饰后，DBM/LN 能黏附更多的 EPCs （ P<0.05），且增殖速度增加、管腔形成能力增强。Western blot 检测示 DBM/LN 中 EPCs 表达 VEGF、p-FAK 和 p-ERK1/2 水平显著高于 DBM（ P<0.05）。动物实验观测显示，与 DBM 组相比，DBM/LN 组骨缺损处血管生成和骨再生能力更强，血管体积和血管表面积差异均有统计学意义（ P<0.05），CD31 hiEmcn hi细胞数量显著增加（ P<0.05）。.
结论: DBM/LN 支架材料能促进 EPCs 黏附、H 型血管生成，实现小鼠骨缺损修复中血管生成和骨再生的有效耦联。.
Keywords: H-type vessels; Laminin α4 chains; bone regeneration; demineralized bone matrix; endothelial progenitor cells; mouse.